In the context of the search for active molecules, and in particular for new antibiotics, amino acids, enzymes and vitamins, various methods are proposed, the principle of which is based on screening the metabolites produced by organisms originating from various environments. Thus, highly important products with antibiotic, anticancer or pesticidal activities have been isolated from microorganisms such as, for example, Streptomyces, Nocardia or Actinoplanes, etc.
Soil and sediments constitute major environments for searching for new active metabolites, not only by virtue of the very large amount of microorganisms which they contain, but also due to the considerable diversity of these microorganisms. It is known that the soil can contain from 109 to 1011 bacteria per gram, of which a maximum of 1% are cultivatable on synthetic media (see R. I. AMANN, W. Ludwig, K H Schleifer (1995) “Phylogenetic identification and in situ detection of individual microbial cells without cultivate” Microbiological Review vol. 59 No. 11 143–169; W B Whitman, D C Coleman, W J Wiebe, 1998. Prokaryotes: The unseen majority, Proceeding National Academy of Science USA, Vol. 95: 6578–6583).
Moreover, the number of bacterial species per gram of soil is evaluated at 1,000 to 10,000. In fact, with DNA—DNA reassociation techniques, the results indicate that the genetic diversity of the microorganisms is certainly greater than 4 000 species per soil sample (Torsvik et al., 1990, Applied Environmental Microbiology, 56: 782–787). Still based on methods which do not involve culturing microorganisms in vitro, V. Torsvik, J. Goksoyr, F L. Daae, R. Sorheim, J. Michalsen and K. Salte, 1994, p. 39–48, in Beyond the Biomass, K. Ritz, J. Dighton and K. E. Giller (eds.), John Wiley and Sons, Chichester, indicate that the diversity of bacterial species may reach 13 000 species per 100 g of soil. Thus, estimating there to be 1010 bacterial cells per gram of soil, a bacterial species should be represented by an average of 106 cells, even for the rarest species.
Moreover, bacteria have been detected in a large number of environments ranging from the stratosphere to the very depths of abysses, including the most extreme media in terms of physicochemical conditions. Thus, bacterial diversity is explained by a diversity of location of bacterial populations, first of all in terms of the macroenvironments which they have colonized, but also in terms of the microenvironments which characterize, for example, and in a nonlimiting manner, the structuring of soils, as described by L. Ranjar, F. Poly, J. Combrisson, A. Richaume, S. Nazaret, 1998. A single procedure to recover DNA from the surface or inside aggregates and in various fractions of soil suitable for PCR-based assays of bacterial communities. European Journal of Soil Biology, 34(2), 89–97.
A large number of studies relating to bacterial diversity, based on the analysis of 16S rDNA ribosomal DNA genes, reveal the presence of many new phyla belonging to the domains of Archaebacteria, but also bacteria. The number of bacterial divisions identified has tripled in 10 years. Thus, the vast diversity of soil bacteria is still unknown. It is largely ignored in scientific research study and is not accessible and therefore not exploited industrially.
One of the objectives pursued is to gain access to this vast potential by extracting the DNA from the 99% of remaining non-cultivatable bacteria.
To do this, a first solution consists in improving culture media by increasing knowledge regarding the physiology of bacteria so as to make them cultivatable and therefore decrease the number of non-cultivatable bacteria. Such a technique has been used since the beginnings of microbiology and has produced very good results. In fact, close to 5,000 microorganisms have been described, all environments taken into account. Thus, more than 40,000 molecules have been characterized and close to half have biological activity. However, such a technique has the drawback of being relatively slow and tedious.
Another solution consists in directly extracting the DNA of non-cultivatable organisms from various environments by chemical and/or enzymatic lysis, as described, for example, in documents U.S. Pat. No. 5,824,485 or WO 97/12991, or else Antonia Suau et al., “Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut” APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 65, No. 11, November 1999, pages 4799 and 4807, and P. S. ROCHELLE et al., “A simple technique for electroelution of DNA from environmental samples” BIOTECHNIQUES, Vol. 11, No. 6, 1991, pages 724, 726–728. The environments considered are either of the aquatic type, which requires concentration of the bacterial cells, as illustrated by the works of J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya and E. deLong, 1996—Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon, Journal of Bacteriology, 178: 591–599, or of the terrestrial type.
One of the main drawbacks of this direct extraction technique is that it leads to the extraction of only DNA which is small in size, of the order of a maximum of 1 to 23 kB. In fact, the physicochemical constraints which are imposed on the DNA during this type of experiment lead to its degradation. A. Frostegard, S. Courtois, V. Ramisse, S. Clerc, D. Bernillon, F. Le Gall, P. Jeannin, X. Nesme, P. Simonet, 1999. Quantification of bias related to the extraction of DNA directly from soils. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, VOL. 65 (12): 5409–5420, clearly show that the improvement of DNA extraction yields, in particular by grinding or sonication techniques, leads to great degradation of the DNA recovered.
In addition, the extracted DNA comprises the extracellular DNA contained in the soil, but also the eukaryotic (fungi, plant cells, animal cells) and prokaryotic (bacteria) DNA and the other organisms present in the soil (protozoa, etc.). It results therefrom that the DNA libraries obtained are often highly contaminated with recombinant bacterial clones containing undesired DNA (eukaryotic DNA, degraded extracellular DNA). Moreover, the libraries thus constituted are characterized by DNA inserts which are small in size (less than 30 Kb). These libraries are not therefore suitable for applications such as the analysis of complete genomes or metagenomes, or the search for, study and exploitation of complete or virtually complete metabolic pathways.
Due to the dilution of the target DNAs by the non-target DNAs, it is consequently essential to selectively amplify the target DNA proportion by PCR so as to constitute appropriate DNA libraries, as described in the abovementioned Suau document. This approach has the main constraint of being able to access only genetic information similar to that already known, in fact excluding access to completely new and original DNA sequences. However, the use of defined PCR primers in very conserved regions has made it possible to isolate genes of interest belonging to nonisolated bacteria, as described, for example, by K. Seow, G. Meurer, M. Gerlitz, E. Wendt-Pienkowski, C. R. Hutchinson and J. Davies, 1997—A study of iterative type II Polyketide Synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms, Journal of Bacteriology, 179: 7360–7368.
Other selection methods have been developed. These methods consist in subjecting the bacterial community to a selection pressure making it possible to enrich given bacterial populations. It is thus hoped to have preferential access to the genetic information desired. Other selections based on the DNA composition (% GC) or its complexity have also been described, such as, for example, document WO 99/45154.